Nanotechnology Flashcards
Discuss why mixing can be a problem in microfluidic devices and its solutions.
Since channels in microfluidics are small (~5-10 μm wide) Reynolds number becomes < 1000, so the motion of the fluid takes place without turbulence, or in a deterministic and reversible way. In this case, the motion of the fluid is called “laminar”: laminar flow is obtained in small channels, or when viscosity is large. The fluid moves as if it were made of sheets that flow one on the other and the velocity of flow varies from zero at the walls to a maximum along the cross-sectional center of the channel. Without turbulence, diffusion is the only way to mix solutions. To achieve mixing has been introduced mixers: by using either mechanical indentations along the channel or electric fields, the fluid (composed of two liquids to be mixed) is put in a rotatory (nonturbulent)
motion, so that the interface between the two components grows and the two liquids become alternated in thin stripes, which become mixed by diffusion.
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According to the Reynold Number equation(RE=densityxUXL/viscosity) that gives the threshold between laminar(<1000) and turbulence(>1000) flows, in microfluidics, we have a laminar motion. In laminar flow, the fluid particles flow in layers adjacent to one another without mixing or only a little mixing due to diffusion. In some cases, the absence of mixing can be exploited for example in selecting motile unicellular or local lysing, but in general, mixing is required in most experimental assessments. To achieve this, the solution is to increase the interfaces between the different components by making thin stripes in which diffusion is efficient and fast. You can create these patterns by using either mechanical indentations along the channel (lamellar structure) or electric fields (stripy pattern) to put the fluids to mix in rotatory motion.
Describe the main features of the two families of microfluidic devices
We have two basic microfluidics technologies:
- Continuous flow: rely on the control of a steady-state liquid flow through narrow channels or porous media predominantly by accelerating or hindering fluid flow in capillary elements. The sample continuously flows in the channels. The actuation of liquid flow is implemented either by external pressure sources, external mechanical pumps, pumps, or by combinations of capillary forces and electrokinetic mechanisms. This mechanism is the less sensitive to protein fouling problems: if we have a fluid of a solution of proteins and on the other side a flow of a solution of denaturant that has come together they mix so we can measure a certain distance in which the proteins denature to see how fast they do it.
- ## Droplet microfluidics: manipulates discrete volumes of fluids in immiscible phases with low Reynolds number and laminar flow regimes. The sample is divided into droplets that don’t mix at all with the water and that can be fused with others or divided. So we can distinct solutions to manipulate the fluid within the channel. Control channels: we have some fluids and they have separated thanks to a flexible membrane from the other channels that are closed by making pressure on the control channel that invades them (the control channel goes over the other channel).There are two different types of microfluidics:
- Continuous flow, in which the flow forks, mix, and go. It’s a continuous flow of one single fluid.
A microfluidic device is made of tubes, valves, and pumps. Usually, the circuits contain two layers, with a thin impermeable flexible membrane between them. One circuit is used to perform experiments and the other one is the control; the channels in the control circuit enable to close off the channels in the main circuit by everting the control channel in the main channel, exploiting the elasticity of the thin rubber membrane separating them. So, putting pressure on the control channel, the flexible membrane will inflate in the channel of interest, invading and closing it. Using this method is also possible to make a pump, using three control channels.
Another feature of continuous flow is mixing. Talking about microfluidics it’s important to consider that it follows a laminar motion, so, having a microfluidic circuit and two fluids, they won’t mix but will run side by side. It is possible to mix efficiently, using either mechanical indentations along the channel or electric fields; in this way, the fluid (composed of two liquids to be mixed) is put in a rotatory (nonturbulent) motion, so that the interface between the two components grows and the two liquids become alternated in thin stripes, which become mixed by diffusion.
Added to diffusion, another phenomenon occurs dispersion. This is a crucial issue in large fluidic circuits, but the situation is less problematic in microfluidics. The combination of diffusion and dispersion causes the diffusion of both the fluid that is left along the walls and the one at the center of the channel. Consequently, the fluid close to the walls moves on average toward the center where the speed is larger, while the contrary is true for the fluid at the center of the channel. When channels are small, this diffusive motion is fast and thus a segment of a given fluid within a channel moves forward with the minimal amount left on the walls. - Droplet microfluidics, in which there is oil, typically a fluorinated oil (which doesn’t mix with water), and droplets inside it. Each droplet is like a single chamber/reactor, so it is possible to operate separately on each one.
Also, in this case, there is no turbulent motion, and it is possible to build circuits in different ways. For example, loading, meaning that an aqueous solution containing solute, cells, or NPs is broken into tiny droplets; or formulating, using different fluids which won’t mix perfectly but will stay side by side in the droplets.
Thanks to this type of microfluidics, is also possible to control and manipulate the droplets, and consequently what is inside them, applying electric fields.
Describe an example of use of a microfluidic device.
An example of a microfluidic device is the PCR on a chip. PCR reactants (DNA to amplify, primers, Mg2+, Taq Pol, salts, and dNTPs) are loaded in a channel. Then the flow brings reactants through three healed circuit compartments, which occur chronologically denaturation, annealing, and elongation, and this cycle is repeated n times depending on how many times the sample has to be amplified. Denaturation: this step is carried out at 94-98°C and causes the separation of the DNA strands. Annealing: this step is carried out at 50-65°C to allow the annealing of the primers to the single-stranded DNA templates at the specific locations corresponding to the two extremities of the specific amplified DNA sequence. Elongation: this step is usually carried out at around 70°C to allow Taq Pol to polymerase complemental DNA strands using the sample DNA strands, separated during the denaturation phase, as templates. As in thermocycler, the time in which reactants stay at a certain temperature is finely regulated. Since the three compartments are maintained at constant temperatures, the time of denaturation, annealing, and elongation processes is determined by the length of their respective compartments. Quantitative PCR can also be performed by using fluorescent probes or dyes and detecting the intensity at the end of each cycle.
Microfluidic Sanger sequencing is alab-on-a-chipapplication for DNA sequencing, in which the Sanger sequencing steps (thermal cycling, sample purification, and capillary electrophoresis) are integrated on a wafer-scale chip using nanoliter-scale sample volumes.We have a circular hybrid glass-polydimethylsiloxane (PDMS) composed of two layers, one has the control and the other has the channels, in between there is a flexible membrane. The top two glass wafers are thermally bonded and then assembled with a featureless PDMS membrane and manifold wafer.
First of all, we have a reaction chamber, and after the load of the sample we can heat and cool the membrane for the amplification, the next step will be to flow what we amplified in another chamber.
After this, we have a thing similar to an electrophoresis migration: when the electrodes are turned on the nucleic acids start running down and they are captured by beats where we have the complementary primers. When the electrodes are turned on the nucleic acids start running down and they are captured by beats where we have the complementary primers. The nucleic acids are captured where we can see them (i.e.) the yellow line and all the rest that we don’t care about goes down.
Microfluidics can be used in PCR, having the channel moving in regions of the circuit that are kept at different temperatures. So, it will follow the melting, the annealing, and then the elongation. The length of the path within the region of a given temperature is the time the fluid stays there. (figure)
Another application of microfluidics can be seen in optimizing synthetic efficiency, particularly in preparing sensitive compounds. A study achieved the synthesis of a fluoride-radiolabeled molecular imaging probe in an integrated microfluidic device. They used a five sequential processes circuit: (I)concentration of dilute fluoride ion with the use of miniaturized anion exchange column, (II) solvent exchange from water to MeCN, (III) fluorination of the D-mannose triflate precursor, (IV) solvent exchange back to water and (V) acidic hydrolysis of the fluorinated intermediate.
Finally, a third example can be the use of droplet microfluidics for enzyme profiling and screening. To screen pharmaceutical leads, modules precisely combine drug library compounds with the enzyme target and then mix and incubate the reactants with an assay substrate in each microdroplet. Each one of them is tagged using a proprietary liquid labeling technique, allowing quantitative experiments and online statistical analysis. Stable, heterogeneous libraries of labeled leads and targets can be prepared to quickly screen all possible combinations.
Describe what is measured in depolarized fluorescence
In depolarized fluorescence, two characteristics are measured: the lifetime of the fluorescent excited state (tF) and the lifetime for rotational diffusion (tD). Because of their anisotropic chemical structure, all fluorophores have a preferential direction of absorption. Each molecule has an axis, such that light polarized along with it, is more efficiently absorbed than light polarized perpendicularly to it. Analogously, emitted fluorescence light is polarized along the same axis. By illuminating a system with polarized excitation light, the fluorophores with their axis along the polarization are more probably excited than those perpendicular to it. Thus, if the fluorophores rotate slowly (fluorescence lifetime larger than rotational diffusion) fluorescent emission is polarized in the same direction as the excitation light. While if it rotates fast (fluorescence lifetime smaller than rotational diffusion) the polarization is lost.
The fluid is illuminated with a polarized excitation light with the use of a linear polarized. Fluorescence emission is measured after having been selected by a second polarizer, that can be parallel (||) or perpendicular (⊥) to one of the excitation lights. The fluorescence intensity measured in the two conditions (I|| and I⊥) can be either the same, meaning that the emission is not polarized (P = 0) or I|| can be larger than I⊥, which indicates polarized fluorescence emission (P > 0).
Describe what information is carried by depolarized fluorescence and how it can be of use.
Because of their anisotropic chemical structure, all fluorophores have a preferential direction of absorption. Each molecule has an axis such that light polarized along it is more efficiently absorbed than light polarized perpendicularly to it. Analogously, emitted fluorescence light is polarized along the same axis.
By illuminating a system with polarized excitation light, the fluorophores with their axis along the polarization are more probably excited than those perpendicular to it.
- If the fluorophores (that bind targets or are analytes themselves) rotate slowly (fluorescence lifetime larger than rotational diffusion) fluorescent emission is polarized in the same direction as the excitation light.
- If they rotate fast (fluorescence lifetime smaller than rotational diffusion) the polarization is lost.
Depolarized fluorescence exploiting the “competition” between fluorescence lifetime and lifetime for rotational diffusion can detect changes in mass within molecules, for example, the formation of Ag and Ab complex. Little molecules have a low Td so they rotate fast while bigger molecules with a high Td rotate slowly. Fluorescent molecules have a preferential direction for absorption (Anisotropic chemical structure), implying that illuminating a sample with polarized light, the fluorophores in the axis with the polarized direction are probably more excited than those perpendicular to it. The combination of the two lifetimes brings to the fact that if TdTf (bigger molecule) the molecules rotates slowly and the polarization is kept. To evaluate this the system is illuminated by polarized light and the light in emission is evaluated in both parallel and perpendicular components. If the two components are equal it means that the polarization is lost, while if the parallel component is larger than the perpendicular it indicates polarized fluorescence emission.
What is “quantum dot” particles? What could they be used for?
Quantum dots are nanoparticles made of semiconductor materials. Although semiconductors are not fluorescent materials when in macroscopic amounts, they become so when cut into nanoparticles. The nanometric size gives some materials new properties.
As the size of the nanoparticles is reduced, the energy gap between the ground state and excited electrons grows and reaches values in the visible range.
Thus, when the electrons of quantum dots are excited, they jump into the excited states (“conduction band”) and they lose energy without emission (by collisions) until they reach the lowest energy in the conduction band. The relaxation to the ground state (“valence band”) releases energy in form of light. So, we can conclude that this fluorescence depends on the size.
They absorb and then emit light. When they do so, even if the dots are made of the same material, the light emitted is in a specific color (or wavelength) depending on the size of its core.
Quantum dots are nanoparticles derived from semiconductors used in visualization for their particular size-dependent properties: semiconductors usually are not fluorescent, but they are when in nano-size. Quantum dots are obtained by reducing their size below the excitation radius. Under the excitation radius, the energy gap between the excited state (conduction band) and the ground state (valence band) increases, therefore the wavelength decreases reaching the visible range. Electrons excited by UV light reach the conduction band and when the electron falls from the excited state to the ground state it emits visible light. The color of the emitted light is size-dependent since energy correlates inversely with energy: little size corresponds to high energy with a blue-violet emission (shorter wavelength) and on the other hand, big size corresponds to low energy, reddish emission (longer wavelength). The emission of different colors is narrow. The use of quantum dots is limited because they are large objects and since they are artificial they can’t be metabolized, they can be used in long resolved detection because they have a long emission lifetime; their main advantage is that they are never photobleached.
Why are magnetic nanoparticles used in magnetic resonance imaging?
Nanomagnetic particles are not magnetic unless there is a magnetic field. However, since they tend to align their spins, their response (susceptibility) to the magnetic field is very large. Small fields induce large magnetization, so nanoparticles of FM materials are superparamagnetic (SPM). In NMR is used a constant magnetic field is to align each hydrogen nucleus’s magnetic momenta and an electromagnetic field having their Larmor frequency (resonance).
- T1: The radiofrequency pulse orients all spins in the XY plane, the positive and negative z components are equal. After the pulse is measured the time in which the spins become again oriented preferentially with the external field
- T2: the time for the spins to become randomly oriented in the XY plane
Magnetic nanoparticles are used to give contrast in MRI due to their high magnetic susceptibility in responding to magnetic fields, becoming readily magnetized. In this way, the surrounding is under the influence of combined magnetic fields: the external magnetic field used in MRI and the one given by the magnetization of the nanomags. The final result is that the magnetic field results are stronger since the nanomags add themselves to it and are less homogenous depending on nanomags position, increasing the T2 sensitivity by shortening it. The use of nanomags impacts also T1. Furthermore, nanomags can be functionalized by bioconjugation to target specif tissues, therefore they can be exploited even as therapeutic tools.
How can the aggregation of gold nanoparticles be measured and exploited for molecular detection?
In metal nanoparticles absorption depends on the particle size, therefore aggregation changes their absorption spectrum since it’s size-dependent. Gold nanoparticles can be used in protease assay to verify the protease activity of a given protein. To achieve this NPs need to be functionalized by coating them with the carboxylic group to make them interact with a given peptide sequence, that’s recognized by protease, giving aggregation. We incubate the peptide sequence with the (supposed) protease and after a while, we add the gold NP in not aggregated forms. If the emission color doesn’t change it means that the protease activity is present, because the cleaved peptide is not able to induce aggregation, on the other hand, if the color emission changes it means that there is no protease activity and that the uncleaved sequence is inducing MNP aggregation.
Describe how the optical properties of nanometallic particles and quantum dots depend on their size.
Nanometallic particles and quantum dots are nanoparticles typically used for their small size and the properties derived from them. Quantum dots are semiconductors meaning that, when excited, the electrons are free to move around the nuclei on different energy levels called conduction bands. When this material is cut down into small pieces (smaller than the exciton radius, the maximum distance from the nucleus which an electron can reach), its proprieties start to change. The result is that the smaller they are, the larger the energy gap between the ground state and the excited state until this energy enters the visible range and changes color. Cutting smaller and smaller, the gap becomes larger and larger: the color emission goes from red to blue. The same thing happens in nanometallic particles. The optical absorption of metal nanoparticles is due to the oscillation of its “surface plasmons”. In metals, one electron per atom is free to move. Surface plasmons are waves of electron density in proximity to the metal surface. These waves have a resonance frequency that depends on the size of the nanoparticle.
What is it meant by bioconjugation of nanoparticles? Make a list of molecules used to bioconjugate nanoparticles and their goal.
Bioconjugation consist of treating surfaces of nanoparticles with biologically relevant molecules is the main way to control the fate of the nanoparticles themselves in solutions. The main reasons to bioconjugate nanoparticles are three:
- Drug delivery: to transport the to the target by using for example lipophilic polymers in water that when collapsed in NP (liposomes) internalize a given drug. When is internalized, after the degradation or the decay of polymers the drug is delivered in solution and can reach the target.
- using them as local probes. i.e. coating with biotin (as a general linker to bind L conjugated with avidin) or antibodies of quantum dots to perform imaging of a given target.
- ## uptake in cells. i.e. Lipid and clathrin coating of an NP to stimulate endocytosis or NPs coated with molecules such as albumin or chitosan that allow crossing the Brain-Blood barrier by adsorptive transcytosis.Bioconjugation, an important element in the use of nanoparticles, is the chemical connection between biologically relevant molecules and the surface of the particle. It is the main way to control the fate of the NP in solutions (e.g. in biological fluids), in cell cultures, tissues, in organisms.
Controlling interactions is crucial in a variety of applications, such as drug delivery, use of NP as local probes, and uptake in cells. The coating of nanoparticles can be generic or specific.
In the first case, the most used is PEG; it is quite soluble, not charged (neutral), hydrophilic, and has a simple and repeated structure. In this case, the NP will not interact with other molecules and won’t be recognized by the immune system. Because of that, this type of coating is used for passive targets.
In the probes with bio affinity, so the specific type, it is possible to find antibody or ligand conjugate. Other examples of bioconjugation can be seen on quantum dots, using a lipid coating, to localize them in a hydrophobic environment, or coating with biotin, as a general linker.
How can magnetic nanoparticles be used to control the local temperature of the tissue hosting them?
Nanomagnetic particles are not magnetic unless there is a magnetic field. However, since they tend to align their spins, their response (susceptibility) to the magnetic field is very large. Small fields induce large magnetization, so nanoparticles of FM materials are superparamagnetic (SPM). Different use of the nanomagnetic particles is considered the affect the temperature (thermotherapy). The application of oscillatory magnetic fields, at the tissue of interest, provokes the increase of the nanomags motion and so generates heat. All the magnetic particles are sensible to the temperature. If you put magnets in a hot environment, they lose their magnetic moment, for example, the proton spin, and so became not magnetic. So, magnetism depends on temperature. The temperature over which the magnetization disappears is called Curie temperature. This tool is a good way to heat the tissues and it is a strategy to enhance locally the temperature, useful for pharmacological strategies that depend on temperature (temperature-controlled drugs). So, oscillatory magnetic fields heat the superparamagnetic
particles. Their Temperature raises because I send energy until they reach the Curie temperature. At larger temperatures, the particles cease to be superparamagnetic and thus the magnetic field loses the capacity of heating them. It is a self-controlled system. There are materials in which, depending on the proportion between iron and aluminum, I can have a Curie temperature that goes from -40° to 280°. So, I can select the material and this is helpful for example to test the efficacy of a specific drug.
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Magnetic nanoparticles can be used for local hyperthermia, which consists of heating locally the temperature of tissues hosting them, this approach can be useful for the localized release of temperature-controlled drugs. The super paramagnetic particles are indeed very sensitive to magnetic fields and their oscillation can heat them by switching the magnetization that leads to losing energy against friction, resulting in heating. Their T raises until they reach the critical temperature (Curie temperature) above which they lose their magnetic properties, therefore they can’t be heated furthermore by the influence of the magnetic field because they can’t respond to it anymore. In this way, the system is self-controlled.
Describe a bioconjugation strategy for the internalization of nanoparticle in cells.
An important example to describe a bioconjugation strategy for the internalization of nanoparticles in the cell is a nanoparticle system that demonstrates an alternative approach to the treatment of cancers, through the inhibition of cell invasion, while serving as magnetic resonance and optical imaging contrast agent. The nanoparticle is comprised of an iron oxide nanoparticle core, conjugated with an amine-functionalized PEG silane and a small peptide, chlorotoxin (CTX), which enables the tumor cell-specific binding of the nanoparticle. the CTX-enabled nanoparticles deactivated the membrane-bound matrix metalloproteinase 2 (MMP-2) and induced increased internalization of lipid rafts that contain surface-expressed MMP-2 and volume-regulating ion channels through receptor-mediated endocytosis, leading to enhanced prohibitory effects. Since upregulation and activity of MMP-2 have been observed in tumors of neuroectodermal origin, and cancers of the breast, colon, skin, lung, prostate, ovaries, and a host of others, this nanoparticle system can be potentially used for non-invasive diagnosis and treatment of a variety of cancer types.
Another example: Receptor-mediated transcytosis is the most common type of transport for NP (nanoparticles) entry into the brain. NPs can be functionalized with different types of ligands (insulin, transferrin, lactoferrin, or antibodies against endothelial receptors), or surfactants (that adsorbs plasma proteins enabling their binding). The interaction between NP ligands and respective receptors triggers the formation of vesicles, which facilitates the release of the NPs on the opposite side of the membrane. NPs coated with molecules such as albumin or chitosan can cross the BBB by adsorptive transcytosis.
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It has been found that NP can be uptaken by various classes of cells, such as endothelium, epithelium, neurons, and macrophages. The uptake which occurs thanks to macrophages is the easiest way and it is generally induced by coating with phosphatidylcholine, signaling apoptosis. For other cell types, the most common strategy is to use clathrin-dependent endocytosis, which is mediated by receptors (the interaction causes the recruiting of clathrin).
Moreover, NP often ends up in lysosomes, which can be a good destination or not depending on the goal of the internalization.
To enter the blood-brain barrier, the receptor-mediated transcytosis is the most common type of transport for NP, which can be functionalized with different types of ligands (such as insulin, transferrin, lactoferrin, or antibodies against endothelial receptors), or surfactants (that adsorbs plasma proteins enabling their binding). The interaction between NP ligands and the respective receptors triggers the formation of vesicles, which facilitates the release of the NPs at the opposite side of the membrane. NPs coated with molecules such as albumin or chitosan can also cross the BBB by adsorptive transcytosis.
Describe the principle of laser tweezing
Optical tweezers use beams of light to hold and manipulate microscopically small objects such as biological molecules or even living cells. They are formed when a laser beam is tightly focused on a tiny region in space using a microscope objective as a lens. This region becomes an optical trap that can hold small objects in 3D. This trapping force that holds an object in place in optical tweezers can be understood by considering how the object refracts light. Because it is tightly focused, the laser light is most intense at the center of the trap, which means that if the object moves slightly away from the center in a transverse direction, one part of the object will refract less light than the other. As a result, the object refracts more light away from the center of the trap than towards it.
Light carries momentum and the net effect of this refraction is a force that deflects some of this momentum away from the center of the trap. By Newton’s third law an equal and opposite force must act on the object, pushing it towards the center of the trap. A similar refraction-related effect also causes the object to push back in the opposite direction of the laser beam. The trapping is stable only if the force of the laser light scattering from the particle along the positive z-direction is compensated by a trapping force along the negative z-direction. To achieve this, a very tight focus is needed, with a significant fraction of the incident light coming in from large angles. This can be achieved using a lens with a high numerical aperture. Force-sensing optical tweezers have the additional ability to track the motion of an object within the trap, information that can then be used to calculate any external forces acting on the object. External forces tend to displace the object from the center of the trap. Using interferometry measurements of the light refracted from the object, this displacement can be determined to nanometer accuracy, which allows the external forces to be measured at the sub-piconewton level. Such external forces depend on the viscosity of the solvent and the properties of the trapped object. In addition, the trapped object can be pushed or pulled on other objects and the forces involved can be measured.
Since their invention, optical tweezers have been used with great success in the field of single-molecule biophysics. For example, they have helped researchers unravel the complex elasticity and folding dynamics of DNA, RNA, and proteins.
Optical tweezers have also helped further our understanding of how “motor proteins” such as kinesin and myosin convert chemical energy into work. Such biological motors operate over distances of nanometers and with picoNewton forces — and the desire to understand motor proteins has been an important driver in the development of force-sensing optical tweezers technology.
Describe how laser tweezers can be used to measure biomolecular properties and which quantities can be determined.
Since the bead deflects light, light exerts a force on the particle. Such force is directed to bring the particle back to the axis. The divergence of the beam changes, which gives rise to forces that push (or pull) the particle toward the waist. The position of the particle can be deduced by the direction and the width of the beam after it has crossed the particle. Thus, at any moment it is possible to know the position in 3D and the force, in 3D on the particle. We could use laser tweezers (LT) to measure biomolecular properties, for example using LT to touch cells with particles conjugated with the potential ligand. So, for studying the interaction between a virus-coated microsphere and the red blood cell, this interaction depends on the presence of the inhibitors. So with laser tweezers, you have a violet light if the inhibitors are absent and the interaction between virus coated microsphere and the red blood cells happens, while in the presence of inhibitors you have a green light because you have an interaction between them.
Another example: Exploiting tweezers’ properties example can study the folding/unfolding process of a protein. For example, using a piezoelectric stage to maintain a nanoparticle conjugated with a linker DNA (that can bind the protein) in a given position and by moving away or bringing closer another nanoparticle conjugated with a linker DNA using the beam as an “optical trap” it’s possible to unfold/fold progressively the protein and to measure the force implied.
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Laser tweezers are a technique that uses the force light can put on a particle to measure biomolecular properties; if the particle is centered in the beam waist (the center of the particle coincides with the point of the minimum size of the beam), its presence does not modify the propagation of light and no force acts on the particle. However, if you move the particle, it will deflect light: it will be like the particle has put a force on light and consequently, you will have a reaction force from light. This is useful because:
- The particle has a lens effect, the light that is reflected tells us where and what the particle is both in 2D and in 3D.
- The reaction force can move nanoparticles and I can use this to measure binding forces, characterize the strength of a motor protein or the force needed to denature a protein quantitatively: e.g. I can use a nanoparticle coated with a ligand, and use laser tweezers I can push cells and particle together and then pull them back until they break free.
Describe what it is meant by label-free biosensors and how they differ from ELISA essays.
Biosensors are analytical devices in which a biological element interacts with analytes (for example antibodies with antigens, receptors with ligands, and so on) and this is coupled to an optical or electronic component, the transducer, which is capable to transform the interaction between the biological element and the analytes into an optical or electric signal. The ELISA assay is a test in which antibodies and colour changing markers are used to identify the amount of a substance. We have a sample with an unknown amount of antigen immobilized on a solid support and any antibodies for the disease I’m testing for will bind to the antigens. Next, a second antibody with a marker is added and a positive reaction is detected by the marker changing colour when an appropriate substrate is added. Thus, I receive the signal that I can measure.
On the contrary, in label-free detection, the interaction itself generates a signal which indicates the interaction, thanks to the capability to induce a physical property modification that can be measured. label-free biosensors are less sensitive than other technologies based on labelled amplification, but they are highly convenient when ligands are not perfectly known when screening a large variety of molecules, and when real-time binding detection is important.
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A biosensor is an analytical device composed of a biological element (e.g enzyme, receptor, antibody) and a physicochemical transducer. Interaction between the target analyte and the biological element produces a change detected by the transducer, which yields a signal we can detect and that it tells us e.g. if the target is present, its concentration or the kinetics of the interaction. Label-free biosensors are those that detect the interaction without using fluorescent or radioactive markers and they are useful if you don’t know what molecule you’re studying. They differ from the ELISA essay because in the latter we use label-based biosensors, which means that we have a fluorescent tag linked to a secondary antibody which detects the primary antibody that is linked to the antigen of interest.
